The proton-ATPase enzyme is an electrogenic proton pump found in mitochondrial membranes, in chloroplast membranes and in bacterial plasma membranes. Many workers now believe that this ATPase is a universal and fundamental feature of the living cell. Its function is to catalyse ATP synthesis and to catalyse active transport of nutrients and ions. The elucidation of the mechanism of action of this enzyme is the goal of our research. There is firm experimental evidence that this enzyme can bind ATP and ADP with extremely high affinity. Several workers have hypothesized that this tightly-bound nucleotide is involved directly in energy-transduction catalysed by the enzyme. There is also evidence (less firm) that Mg ions bind to the enzyme, although the nature of these binding sites is not well understood. However in proposing mechanisms of ATP hydrolysis and synthesis, many workers have invoked a role for Mg. It is the purpose of this proposal to thoroughly characterise the binding sites for tightly-bound nucleotide and to investigate what kinds of sites exist for binding of magnesium. In the first part of the work membrane reconstitution procedures and new procedures developed here and elsewhere for removing and reloading the tightly-bound nucleotides will be employed to define the number of sites and the structural specificity of the sites on the membrane-bound enzyme. Effects of ATP and ADP analogues (inhibitors, other substrates) incorporated in these sites will be studied. Effects of energisation, inhibition of energy transfer and uncouplers will be studied. Reactive ATP and ADP analogues will be utilized to label the sites and allow localisation of them on the enzyme subunits. The aim of the first part of the work is to define the role of the tightly-bound nucleotide in energy transduction. In the second part of the work the number and nature of Mg sites will be established.